1. Field
This application relates to diagnostics, assays and detection reagents. In particular, the application describes reagents that are able to detect and distinguish monomers, oligomers, and polymers of ADP-ribose. These reagents are constructed to have the advantageous properties of antibodies with different specificity, selectivity, and affinity than has previously been reported for anti-ADP-ribose antibodies (e.g., the 10H monoclonal antibody).
2. Related Art
ADP-ribose is a naturally-occurring small molecule with a variety of functions. It is commonly found linked to proteins as a post-translational modification. Mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) transferase enzymes catalyse the transfer (and in the case of PAR transferase enzymes, polymerization) of ADP-ribose units from NAD+, which can be covalently linked glutamate, aspartate, and lysine residues of acceptor proteins. DNA-strand breakage has been considered the main trigger of MAR and PAR synthesis, leading either to repair of the damaged site and cell survival, or cell death, depending on the cellular context and on the intensity of the DNA insult. However, other cellular components (e.g., interacting proteins, nucleosomes, posttranslational modifications, etc.) may also stimulate MAR and PAR synthesis and the size and branching of PAR synthesized under normal conditions is still unclear.
There are at present 17 PARP family members, and it remains to be determined whether all PARP family members can effectively synthesize MAR and/or PAR and, if so, whether they produce structures comparable to that synthesized by the founding member PARP1. Some PARP family members lack conserved residues crucial for polymer elongation and may instead be mono(ADP-ribose) transferases. A detailed biochemical characterization of each PARP family member is necessary to answer the numerous questions that remain regarding PAR synthesis, transfer, function and degradation.
At present, the most widely used (and possibly only available) reagent to investigate ADP-ribose utilization is a monoclonal antibody against poly(ADP-ribose) (PAR). A major drawback of this reagent is that it only detects ADP-ribose in chains of more than ˜10 units. In many biological systems, monomers or oligomers of ADP-ribose are the most relevant form.